CN108641961B - Method for high-density culture of guava leaf endophytes - Google Patents
Method for high-density culture of guava leaf endophytes Download PDFInfo
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- CN108641961B CN108641961B CN201810326770.3A CN201810326770A CN108641961B CN 108641961 B CN108641961 B CN 108641961B CN 201810326770 A CN201810326770 A CN 201810326770A CN 108641961 B CN108641961 B CN 108641961B
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Abstract
A method for culturing guava leaf endophytes at high density comprises the following steps: (1) drying, powdering and sterilizing guava leaves to obtain pretreated guava leaf powder; (2) activating endophytes; (3) inoculating the activated endophyte into a solid culture medium containing the pretreated guava leaf powder obtained in the step (1) for culture to obtain a co-culture product; (4) inoculating the co-culture product obtained in the step (3) into a liquid culture medium to culture to obtain an endophyte seed solution; (5) and (4) adding the endophyte seed liquid obtained in the step (4) and a liquid culture medium containing a phosphorus source into a fermentation tank for high-density culture to obtain high-density guava leaf endophytes. According to the invention, endophyte is cultured at high density in the environment of guava leaves, and a large amount of metabolites with biological activity can be produced through metabolism.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for culturing guava leaf endophytes.
Background
Guava is one of the most popular high-quality fruits in south China, and has good development prospect. The guava biological fruit is fresh, sweet and tasty, fresh, sweet and juicy, fragrant and pleasant, has peculiar flavor and rich nutrient components, and is popular with consumers. In addition, the guava leaves have certain medicinal value, and the guava leaves are widely applied to treating diseases such as diabetes, diarrhea and the like in China and abroad, because the guava leaves contain a plurality of phenolic components with novel structures, and a large number of researches show that the phenolic components of the guava leaves have the effects of reducing blood sugar, resisting oxidation, resisting cancer and resisting pathogenic microorganisms. However, the prior extraction process is not mature, so that the bioactive substances can not be effectively extracted from the guava leaves, and the production cost is high.
Endophytes generally refer to a class of microorganisms (mainly fungi and bacteria) that spend all or nearly all of their life cycle in a healthy plant host without causing the host to exhibit any symptoms, and that live inside various tissues and organs of plants, are a natural component of the ecosystem within plants. In the long-term co-evolution process, the endophyte and the plant form a reciprocal and mutual profit relationship. Research shows that in the co-evolution process of endophyte and plant, the endophyte can not only produce special secondary metabolites, but also induce the synthesis of the secondary metabolites of the host plant. The endophyte can also promote the growth of plants, increase the total biomass of the plants, improve the vitality of plant seeds and promote the survival and tillering growth of seedlings by secreting plant hormones or promoting the secretion of the plant hormones, thereby improving the yield of medicinal plants and increasing secondary metabolites.
Therefore, the method for producing the guava metabolite with biological activity by using the endophyte has certain application prospect. Although many endophyte strains that produce secondary metabolites of plants have been isolated, their use in production is almost nil. This is mainly because the content of secondary metabolites synthesized by the in vitro culture of endophytes is low, and it is only possible to obtain high yields of secondary metabolites by large-scale high-density culture of endophytes.
Therefore, how to lead the endophyte to propagate massively and rapidly in vitro is a key problem and is a precondition for realizing the industrial production of secondary metabolites by replacing medicinal plants with the endophyte.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings in the background technology, and provide a guava leaf endophyte culture method capable of realizing large-scale and rapid propagation of endophytes. In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for culturing guava leaf endophytes at high density comprises the following steps:
(1) drying, powdering and sterilizing guava leaves to obtain pretreated guava leaf powder;
(2) inoculating endophyte (preferably endophyte capable of producing terpenoid substances separated and screened from Guangzhou guava leaves) separated and screened from guava leaves to a solid microbial culture medium for endophyte culture activation;
(3) inoculating the activated endophyte into a solid culture medium containing the pretreated guava leaf powder obtained in the step (1) for culture to obtain a co-culture product;
(4) inoculating the co-culture product obtained in the step (3) into a liquid culture medium to culture to obtain an endophyte seed solution;
(5) and (4) adding the endophyte seed liquid obtained in the step (4) and a liquid culture medium containing a phosphorus source into a fermentation tank for high-density culture to obtain high-density guava leaf endophytes.
In the above method for culturing the guava leaf endophyte at high density, it is preferable that,in the step (2), the solid microbial culture medium comprises the following components: 5-7.5g of peptone, 1.5-3g of yeast powder, 5-7g of glucose, 3.8-4.5mg of vitamin complex, 1-2.3mL of trace salt solution, 15g of agar and 1L of sterile water; the vitamin complex comprises the following components in parts by weight: 1 part of vitamin A, 1 part of vitamin B1, 1 part of vitamin B2, 0.5 part of vitamin PP and 0.5 part of tea polyphenol; the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, 100mL of sterile water. The effect of adding a trace amount of salt solution to the culture medium is as follows: NaCl can balance osmotic pressure and plays a role in regulating the osmotic pressure in a culture medium; mg (magnesium)2+Is an important enzyme activator for EMP and TCA ways and lysine generation, and forms the active groups of cytochrome, cytochrome oxidase and catalase; fe2+Is one of the important members of the electronic respiratory transmission chain. In addition, the solid microorganism culture medium has the following advantages: 1. the carbon/nitrogen source content is reasonable, which is beneficial to the growth of endophytes; 2. contains specific factor vitamin complex, which can regulate the metabolism of microbe and has cell factor stimulating cell growth activity to shorten the activation time. The activation time of the endophyte can be shortened by 12-18h by controlling the reasonable content of the carbon/nitrogen source and adding a specific factor.
In the method for culturing the guava leaf endophyte at high density, preferably, the process conditions for culturing and activating the endophyte in the step (2) are as follows: the proportion relation between the inoculation amount and the carbon source and the nitrogen source in the solid microorganism culture medium is as follows: inoculation amount (10)7cells): carbon source (g): the nitrogen source (g) is 1.6:4-6:1-1.5, more preferably 1.6:5:1.3, and the culture temperature is controlled at 27-30 deg.C for 20-36 h. The carbon source and the nitrogen source are nutrients essential for the growth of the microorganism, and the ratio of the inoculation amount to the carbon/nitrogen source is related to the growth and propagation process of endophytes. If the carbon source amount is too much, a lower pH value environment is easily formed, the growth of thalli is not facilitated, and if the carbon source amount is insufficient, the thalli is easy to age and autolyze; if the amount of nitrogen source is too large, the growth of the cells will be too vigorous, and higher growth will occurThe pH value environment is not beneficial to the accumulation of metabolites, and if the quantity of the nitrogen source is insufficient, the reproduction quantity of thalli is small, so that the yield is influenced; in addition, improper carbon-nitrogen ratio can also affect the proportional absorption of nutrients by the bacteria, directly affecting the growth of the bacteria and the formation of products. A large number of experiments show that only by controlling the inoculation and the carbon/nitrogen source to be in the proportional relation, an environment which is beneficial to the rapid propagation and activation of thalli can be provided. The above-mentioned activation conditions are optimum growth conditions for the cells, and under these conditions, the activity of the cells is good and the cell concentration is highest in the growth stabilizer stage.
In the method for culturing the guava leaf endophytes at high density, in the step (3), the solid medium preferably comprises the following components: 4-6g of peptone, 1.5-3g of yeast powder, 4-6g of glucose, 5-7.5mg of pretreated guava leaf powder, 1-2.3mL of trace salt solution, 15g of agar and 1L of sterile water; wherein the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, 100mL of sterile water. The endophyte is screened from the guava leaves, and the solid culture medium added with the pretreated guava leaf powder in the step (3) is used for providing a guava leaf environment so as to be beneficial to the growth and metabolism of the endophyte; on the other hand, the present invention is considered based on the object of preparing a biologically active substance by co-culturing endophyte with guava leaves, so that pre-treatment of guava leaves is added to the culture medium.
In the method for culturing the guava leaf endophyte at high density, preferably, the process conditions for culturing and obtaining the co-culture product in the step (3) are as follows: the proportion relation between the inoculation amount and the carbon source and the nitrogen source in the solid culture medium is controlled as follows: inoculation amount (10)7cells): carbon source (g): the nitrogen source (g) is 2:4-6:1.4-2.0, more preferably 2:6:1.6, and the culture temperature is controlled to be 27-30 ℃ for 4-7 days. A large number of experiments show that the conditions are the optimal growth conditions of the thalli, under the conditions, the activity of the thalli is better, and the concentration of bacteria is highest in the stable growth period.
In the method for culturing the guava leaf endophyte at high density, it is preferable that in the step (4), the solution isThe composition of the body culture medium comprises: 4-6g of peptone, 1.5-3g of yeast powder, 4-6g of glucose, 5-7.5mg of pretreated guava leaf powder, 1-2.3mL of trace salt solution and 1L of sterile water; wherein the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, 100mL of sterile water.
In the method for culturing the guava leaf endophyte at high density, preferably, the process conditions for obtaining the endophyte seed liquid by culturing in the step (4) are that the initial concentration of the endophyte in the liquid culture medium is controlled to be not less than 1.6 × 107cells/L, and controlling the culture temperature to be 27-30 ℃, the time to be 2-5d, and the rotation speed to be 150-200rpm, more preferably 170 rpm. The above conditions are the optimum growth conditions for the cells, and under these conditions, the activity of the cells is better and the cell concentration is highest in the growth stabilizer stage.
In the method for culturing the guava leaf endophyte at high density, in the step (5), the composition of the liquid culture medium containing the phosphorus source preferably comprises: 4-6g of peptone, 1.5-3g of yeast powder, 4-6g of glucose, 5-7.5mg of pretreated guava leaf powder, 1-2.3mL of trace salt solution and K2HPO40.2 to 0.3g (more preferably 0.25g), 0.10 to 0.20g (more preferably 0.15g) of citric acid diamine, 1L of sterile water; wherein the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, 100mL of sterile water. K2HPO4The addition of the compound fertilizer is to provide a phosphorus source required by the growth and the propagation of endophyte. The citric acid diamine can be used as a buffer solution (together with supplemented ammonia water), so that the adverse effect on the growth of thalli caused by overlarge pH value fluctuation is avoided.
In the method for culturing the guava leaf endophyte at high density, preferably, the process conditions of the high-density culture in the step (5) are as follows: controlling the volume ratio of the endophyte seed liquid to the liquid culture medium containing the phosphorus source to be (0.06-0.1): 1, the initial pH value in the fermenter is 7.0-7.3 (more preferably 7.2), and the dissolved oxygen content (the mass concentration of oxygen in the fermentation broth) is55-65%, the culture temperature is 27-30 ℃, and the rotation speed is 80-100rpm (more preferably 90 rpm); and the liquid culture medium containing the phosphorus source is added into the fermentation tank in a batch feeding mode to control the phosphorus source at the early stage in the fermentation tank to be 0.02-0.05g/L and the phosphorus source at the later stage to be less than 0.0083g/L, wherein the early stage refers to the culture for 5-10h by adding the endophyte seed solution, and the later stage refers to the culture for 4-5d by adding the endophyte seed solution. During high-density culture, phosphorus is needed for growth and propagation of thalli, and endophytes grow in a large quantity in the early stage, so that the phosphorus source quantity is large, the growth of endophytes in the later stage is slow, and the phosphorus concentration is too high, so that the thalli autolysis is easy to cause (the influence of the phosphorus source quantity in the early stage on the thalli autolysis is small, because the growth speed in the early stage of the thalli is far greater than the autolysis speed of the thalli, the autolysis quantity can be ignored), the phosphorus source quantity is small. Control of the amount of phosphorus source the addition of K can be controlled2HPO4The amount of the phosphorus source can be realized by adding the liquid culture medium into the fermentation tank in a batch feeding mode and realizing fed-batch culture to control the amount of the phosphorus source in the fermentation tank. By controlling the phosphorus source and the fermentation culture conditions, the amount of the finally obtained endophyte can be obviously improved.
In the method for high-density culture of the guava leaf endophyte, preferably, during high-density culture, when the pH value in the fermentation tank is reduced to 5.93, ammonia water is supplemented into the fermentation tank to control the pH value in the fermentation tank to be 6.5-7.2, and when the glucose concentration is reduced to 3.5g/L, liquid culture medium without a phosphorus source is started to be supplemented into the fermentation tank to control the glucose concentration in the fermentation tank to be 3.5-6 g/L. The pH value of 7.2 is the optimum growth pH value of the thalli, the pH value of the fermentation liquor can be gradually reduced along with the extension of the culture time, the sugar metabolism is slow due to the overlow pH value, the fermentation time can be prolonged, and when the pH value of the fermentation liquor is reduced to 5.93, ammonia water can be fed into the fermentation tank to control the pH value to be 6.5-7.2 (the thalli are autolyzed due to the overhigh pH value, and the pH value is preferably controlled to be 6.5). In addition, the sugar concentration of the fermentation liquor is gradually reduced along with the prolonging of the culture time, the sugar concentration is too low, the growth and the propagation of the thalli are not facilitated, and the sugar can be supplemented by feeding a liquid culture medium which does not contain a phosphorus source (the generation of phosphorus source fluctuation is avoided, and the growth and the development of the thalli are influenced).
In the method for culturing the guava leaf endophyte at high density, the culture time of the endophyte can be shortened by controlling the components and the proportion of the culture medium and controlling the phosphorus source, the pH value and the sugar concentration in the fermentation tank, the endophyte can also grow in large quantity, and the quantity of the finally obtained endophyte can be increased by more than 6.5 times compared with the common means.
Compared with the prior art, the invention has the advantages that:
1. according to the invention, endophyte is cultured at high density in the environment of guava leaves, and a large amount of metabolites with biological activity can be produced through metabolism.
2. When the endophyte is cultured at high density, the culture time of the endophyte can be greatly shortened and the fungus concentration of the endophyte can be remarkably increased by controlling the components and the proportion of the culture medium.
3. The culture method is simple, easy to operate and suitable for large-scale production and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a process flow diagram of an embodiment of the present invention.
Detailed Description
In order to facilitate an understanding of the invention, the invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1:
as shown in figure 1, a method for culturing guava leaf endophytes at high density comprises the following steps:
(1) pre-treating guava leaves: picking fresh guava leaves of a guava tree, drying the guava leaves at 45 ℃ for 12 hours, then grinding the guava leaves into fine powder, adding sterile water, and filtering the mixture by a filter membrane of 220 mu m to remove other mixed bacteria except specific endophytes;
(2) activation and culture of endophytes: separating and screening endophytes capable of producing terpenoids from guava leaves, inoculating the endophytes into a solid microbial culture medium, and performing endophyte culture activation, wherein the proportion relation between the inoculum size and a carbon source and a nitrogen source in the solid microbial culture medium is controlled as follows: inoculation amount (10)7cells): carbon source (g): the nitrogen source (g) is 1.6:5:1.3, the culture temperature is controlled at 28 ℃, and the culture time is 30 hours; wherein, the solid microorganism culture medium comprises the following components: 6g/L of peptone, 2g/L of yeast powder, 6g/L of glucose, 4.17mg/L of vitamin complex, 1.5mL/L of trace salt solution, 15g/L of agar and sterile water; the vitamin complex solution comprises the following components in parts by weight: 1 part of vitamin A, 1 part of vitamin B1, 1 part of vitamin B2, 0.5 part of vitamin PP and 0.5 part of tea polyphenol; the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water;
(3) co-culturing guava leaves and endophytes in a culture medium: inoculating the activated endophyte into a solid culture medium containing the pretreated guava leaf powder obtained in the step (1) for culture to obtain a co-culture product, wherein the proportional relation between the inoculation amount and a carbon source and a nitrogen source in the solid culture medium is controlled as follows: inoculation amount (10)7cells): carbon source (g): the nitrogen source (g) is 2:6:1.6, the culture temperature is controlled to be 28 ℃, and the culture time is 5 d; wherein, the solid culture medium comprises the following components: 5g/L of peptone, 2g/L of yeast powder, 5g/L of glucose and pretreatment6.5mg/L of pomegranate leaf powder, 1.5mL/L of trace salt solution, 15g/L of agar and sterile water; wherein, the composition of the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water;
(4) inoculating the co-culture product obtained in the step (3) into a liquid culture medium to culture to obtain an endophyte seed liquid, and controlling the initial concentration of endophytes in the liquid culture medium to be 1.6 × 107cells/L, controlling the culture temperature to be 28 ℃, and performing shake culture at 170rpm for 3 d; wherein, the liquid culture medium comprises the following components: 5g/L of peptone, 2g/L of yeast powder, 5g/L of glucose, 6.5mg/L of pretreated guava leaf powder, 1.5mL/L of trace salt solution and sterile water; wherein, the composition of the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water;
(5) adding the endophyte seed liquid obtained in the step (4) and a liquid culture medium containing a phosphorus source into a fermentation tank for high-density culture to obtain high-density guava leaf endophytes, wherein the liquid culture medium containing the phosphorus source comprises the following components: 5g/L of peptone, 2g/L of yeast powder, 5g/L of glucose, 6.5mg/L of pretreated guava leaf powder, 1.5mL/L of trace salt solution and K2HPO40.25g/L, 0.15g/L of citric acid diamine and sterile water; wherein, the composition of the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water; adding a liquid culture medium containing a phosphorus source into a fermentation tank in a batch feeding manner, so as to control the early-stage phosphorus source in the fermentation tank to be 0.0351g/L and the later-stage phosphorus source to be less than 0.0083g/L, control the volume ratio of the seed liquid of the endophytes added into the fermentation tank to the liquid culture medium to be 0.06, control the initial pH value in the fermentation tank to be 7.2, control the dissolved oxygen content to be 55 percent, control the culture temperature to be 28 ℃ and control the rotating speed to be 90 rpm; and during the culture process, when the pH value in the fermentation tank is reduced to 5.93 (generally after 2-3 days of culture), adding ammonia water into the fermentation tank to control the pH value in the fermentation tank6.5, when the glucose concentration is reduced to 3.5g/L (generally after 36h of culture), the liquid culture medium without the phosphorus source is added into the fermentation tank to control the glucose concentration in the fermentation tank to be kept at 4.0g/L and the dissolved oxygen content to be about 50%.
According to the example, 3-4d is logarithmic growth phase of the bacteria, and the tank is placed in the stationary phase of the logarithmic growth, at which time the OD value of the bacteria in the fermentation liquor reaches 2.7, and the total culture time is 4-6 d. Compared with the OD values of the comparative example 1 and the comparative example 2, the OD values of the embodiment are respectively improved by about 7 times and 19 times; the total culture time was shortened by about 1.5 times and 2 times compared with comparative example 1 and comparative example 2, respectively.
Example 2:
as shown in figure 1, a method for culturing guava leaf endophytes at high density comprises the following steps:
(1) pre-treating guava leaves: picking fresh guava leaves of a guava tree, drying the guava leaves for 18h at 45 ℃, then grinding the guava leaves into fine powder, adding sterile water, and filtering the mixture by a filter membrane of 220 mu m to remove other mixed bacteria except specific endophytes;
(2) activation and culture of endophytes: separating and screening endophytes capable of producing terpenoids from guava leaves, inoculating the endophytes into a solid microbial culture medium, and performing endophyte culture activation, wherein the proportion relation between the inoculum size and a carbon source and a nitrogen source in the solid microbial culture medium is controlled as follows: inoculation amount (10)7cells): carbon source (g): the nitrogen source (g) is 1.6:5:1.3, the culture temperature is controlled at 28 ℃, and the culture time is 24 hours; wherein, the solid microorganism culture medium comprises the following components: 6g/L of peptone, 2g/L of yeast powder, 6g/L of glucose, 4.17mg/L of vitamin complex, 1.5mL/L of trace salt solution, 15g/L of agar and sterile water; the vitamin complex solution comprises the following components in parts by weight: 1 part of vitamin A, 1 part of vitamin B1, 1 part of vitamin B2, 0.5 part of vitamin PP and 0.5 part of tea polyphenol; the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water;
(3) co-culturing guava leaves and endophytes in a culture medium: will be activated internallyInoculating the live bacteria into a solid culture medium containing the pretreated guava leaf powder obtained in the step (1) for culture to obtain a co-culture product, wherein the proportional relation between the inoculation amount and a carbon source and a nitrogen source in the solid culture medium is controlled as follows: inoculation amount (10)7cells): carbon source (g): the nitrogen source (g) is 2:6:1.6, the culture temperature is controlled to be 28 ℃, and the culture time is 7 d; wherein, the solid culture medium comprises the following components: 5g/L of peptone, 2g/L of yeast powder, 5g/L of glucose, 6.5mg/L of pretreated guava leaf powder, 1.5mL/L of trace salt solution and sterile water; wherein, the composition of the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water;
(4) inoculating the co-culture product obtained in the step (3) into a liquid culture medium to culture to obtain an endophyte seed liquid, and controlling the initial concentration of endophytes in the liquid culture medium to be 1.6 × 107cells/L, controlling the culture temperature to be 28 ℃, and performing shake culture at 170rpm for 3 d; wherein, the liquid culture medium comprises the following components: 5g/L of peptone, 2g/L of yeast powder, 5g/L of glucose, 6.5mg/L of pretreated guava leaf powder, 1.5mL/L of trace salt solution and sterile water; wherein, the composition of the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water;
(5) adding the endophyte seed liquid obtained in the step (4) and a liquid culture medium containing a phosphorus source into a fermentation tank for high-density culture to obtain high-density guava leaf endophytes, wherein the liquid culture medium containing the phosphorus source comprises the following components: 5g/L of peptone, 2g/L of yeast powder, 5g/L of glucose, 6.5mg/L of pretreated guava leaf powder, 1.5mL/L of trace salt solution and K2HPO40.25g/L, 0.15g/L of citric acid diamine and sterile water; wherein, the composition of the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water; adding liquid culture medium containing phosphorus source into the fermentation tank in batch feeding manner to control the fermentation tankThe phosphorus source at the early stage is 0.0351g/L, the phosphorus source at the later stage is less than 0.0083g/L, the volume ratio of the seed liquid of the endophyte added into the fermentation tank to the liquid culture medium is controlled to be 0.06, the initial pH value in the fermentation tank is controlled to be 7.2, the dissolved oxygen content is 63 percent, the culture temperature is 28 ℃, and the rotation speed is 90 rpm; and in the culture process, when the pH value of the fermentation liquor is reduced to 5.93 (generally after 3 days of culture), adding ammonia water into the fermentation tank to control the pH value in the fermentation tank to be 6.5, and when the glucose concentration is reduced to 3.5g/L (generally after 48 hours of culture), beginning to supplement a liquid culture medium without a phosphorus source into the fermentation tank to control the glucose concentration in the fermentation tank to be kept at 5.5g/L and the content of dissolved oxygen to be about 50 percent.
According to the embodiment, 3-4d is logarithmic growth phase of the thallus, and the thallus is placed in a tank at the stationary phase of the logarithmic growth, wherein the OD value of the thallus in the fermentation liquor reaches 3, and the total culture time is 4-5 d.
Comparative example 1:
this comparative example is different from example 1 in that no vitamin complex is added to the solid microorganism medium in the activation and culture of the endophyte in step (2).
According to the comparative example, 5-8 days are logarithmic growth phase of thallus, and the thallus is placed in a tank at the stationary phase of logarithmic growth, wherein the OD value of the fermentation liquor reaches 0.386, and the total culture time is 7-12 days.
Comparative example 2:
compared with example 1, the difference of this comparative example is that when endophyte is cultured at high density in step (5), phosphorus source K is not added into liquid culture medium2HPO4。
According to the comparative example, 6-8d is the logarithmic growth phase of the thallus, and the tank is placed in the logarithmic growth stationary phase, wherein the OD value of the fermentation liquor reaches 0.142, and the total culture time is 8-13 d.
Comparative example 3:
compared with the embodiment 1, the difference of the comparison example is that the ratio relation between the controlled inoculation amount and the carbon source and the nitrogen source in the solid microorganism culture medium is as follows: inoculation amount (10)7cells): carbon source (g): nitrogen source (g) ═ 1.6:7: 0.8.
According to the comparative example, 5-8 days are logarithmic growth phase of thallus, and the thallus is placed in a tank at the stationary phase of logarithmic growth, wherein the OD value of the fermentation liquor reaches 1.1, and the total culture time is 6-9 days.
Claims (9)
1. A method for culturing guava leaf endophytes at high density is characterized by comprising the following steps:
(1) drying, powdering and sterilizing guava leaves to obtain pretreated guava leaf powder;
(2) inoculating endophyte separated and screened from guava leaves to a solid microbial culture medium for endophyte culture activation; the endophyte is capable of metabolizing to produce terpenoid; the solid microorganism culture medium comprises compound vitamins, and the compound vitamins comprise the following components in parts by weight: 1 part of vitamin A, 1 part of vitamin B1, 1 part of vitamin B2, 0.5 part of vitamin PP and 0.5 part of tea polyphenol;
(3) inoculating the activated endophyte into a solid culture medium containing the pretreated guava leaf powder obtained in the step (1) for culture to obtain a co-culture product;
(4) inoculating the co-culture product obtained in the step (3) into a liquid culture medium to culture to obtain an endophyte seed solution;
(5) adding the endophyte seed liquid obtained in the step (4) and a liquid culture medium containing a phosphorus source into a fermentation tank for high-density culture to obtain high-density guava leaf endophytes;
the ratio of the inoculation amount in the step (2) to the carbon source and the nitrogen source in the solid microorganism culture medium is as follows: inoculation amount (10)7cells): carbon source (g): nitrogen source (g) =1.6:4-6: 1-1.5; the proportion relation between the inoculation amount in the step (3) and the carbon source and the nitrogen source in the solid culture medium is controlled as follows: inoculation amount (10)7cells): carbon source (g): nitrogen source (g) =2:4-6: 1.4-2.0;
the liquid culture medium containing the phosphorus source comprises the following components: 4-6g of peptone, 1.5-3g of yeast powder, 4-6g of glucose, 5-7.5mg of pretreated guava leaf powder, 1-2.3mL of trace salt solution and K2HPO40.2-0.3g, 0.1-0.2g of citric acid diamine and 1L of sterile water; wherein the group of trace salt solutionsThe components of the composition are as follows: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, and 100mL of sterile water; the liquid culture medium containing the phosphorus source is added into the fermentation tank in a batch feeding mode, the phosphorus source at the early stage in the fermentation tank is controlled to be 0.02-0.05g/L, and the phosphorus source at the later stage is controlled to be less than 0.0083g/L, the early stage refers to the culture of the added endophyte seed solution for 5-10h, and the later stage refers to the culture of the added endophyte seed solution for 4-5 d.
2. The method for culturing the guava leaf endophytes at high density according to claim 1, wherein in the step (2), the solid microbial culture medium comprises: 5-7.5g of peptone, 1.5-3g of yeast powder, 5-7g of glucose, 3.8-4.5mg of vitamin complex, 1-2.3mL of trace salt solution, 15g of agar and 1L of sterile water; wherein the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, 100mL of sterile water.
3. The method for high-density culture of the guava leaf endophyte according to claim 1 or 2, wherein the process conditions for culture and activation of the endophyte in the step (2) are as follows: the culture temperature is controlled at 27-30 deg.C, and the culture time is 20-36 h.
4. The method for culturing the guava leaf endophyte at high density according to claim 1, wherein in the step (3), the solid medium comprises the following components: 4-6g of peptone, 1.5-3g of yeast powder, 4-6g of glucose, 5-7.5mg of pretreated guava leaf powder, 1-2.3mL of trace salt solution, 15g of agar and 1L of sterile water; wherein the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, 100mL of sterile water.
5. The method for high-density culture of psidium guajava leaf endophyte according to claim 1 or 4, wherein the process conditions for obtaining the co-culture product by culturing in the step (3) are as follows: controlling the culture temperature at 27-30 deg.C for 4-7 days.
6. The method for culturing the guava leaf endophytes at high density according to claim 1, wherein in the step (4), the composition of the liquid medium comprises: 4-6g of peptone, 1.5-3g of yeast powder, 4-6g of glucose, 5-7.5mg of pretreated guava leaf powder, 1-2.3mL of trace salt solution and 1L of sterile water; wherein the trace salt solution comprises the following components: 0.5g FeSO4·7H2O，0.25g MnCl2·4H2O，0.5g MgSO4·7H2O, 0.5g NaCl, 100mL of sterile water.
7. The method for high-density cultivation of guava leaf endophyte according to claim 1 or 6, wherein the process conditions for obtaining the endophyte seed solution by cultivation in step (4) are that the initial concentration of endophyte in the liquid culture medium is controlled to be not less than 1.6 × 107cells/L, and controlling the culture temperature at 27-30 ℃, the time at 2-5d, and the rotation speed at 150-.
8. The method for high-density culture of psidium guajava leaf endophyte according to claim 1, wherein the process conditions of the high-density culture in the step (5) are as follows: controlling the volume ratio of the endophyte seed liquid to the liquid culture medium containing the phosphorus source to be (0.06-0.1): 1, the initial pH value in the fermentation tank is 7.0-7.3, the dissolved oxygen content is 55-65%, the culture temperature is 27-30 ℃, and the rotation speed is 80-100 rpm.
9. The method according to claim 8, wherein the pH of the fermentor is controlled to 6.5-7.2 by adding ammonia water when the pH value of the fermentor is reduced to 5.93, and the glucose concentration is controlled to 3.5-6g/L by starting to add the liquid medium without the phosphorus source to the fermentor when the glucose concentration is reduced to 3.5 g/L.
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